Copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) has gained widespread use in chemical biology for applications such as labeling of biomolecules in complex mixtures and imaging of fixed cells and tissues. (Kolb, et al., Angew. Chem. Int. Ed. 2001, 40, 2004; Rostovtsev, et al., Angew. Chem. Int. Ed. 2002, 41, 2596; Wu and Fokin, Aldrichimica Acta 2007, 40, 7.) Incorporation of fluorescent probes into proteins, DNA, RNA, lipids and glycans within their native cellular environments provides opportunities for imaging and understanding their roles in vivo. (Best, Biochemistry 2009, 48, 6571.)
For example, glycans in protein are displayed on the cell surface with implications in numerous physiological and pathological processes. Aberrant glycosylation on the surface of diseased cells is often observed in pathological conditions, such as inflammation and cancer metastasis. In particular, altered terminal sialylation and fucosylation, which are believed to result from changes in expression locations and levels of sialyltransferases and fucosyltransferases, are associated with tumor malignancy. The ability to explore the biological information content of glycans as biomarkers of cancer, attached to either proteins or lipids, has become a major course of glycomics research. (Hsu, et al., Proc. Nat. Acad. Sci. U.S.A., 2007, 104, 2614; Sawa, et al., Proc. Nat. Acad. Sci. U.S.A., 2006, 103, 12371.)
Analysis of changes in glycosylation patterns in living systems is now possible. (Prescher and Bertozzi, Nat. Chem. Bio. 2005, 1, 13.) Metabolic incorporation of an unnatural carbohydrate containing unique functional group that acts as a bioorthogonal chemical reporter into the cell biosynthetic machinery initiates the process. The modified glycan is then processed and constructed on the cell surface. Subsequent reaction with a detectable fluorescent probe equipped with a complementary bioorthogonal functional group enables detection of the incorporated unnatural glycan. (Sletten and Bertozzi, Angew. Chem. Int. Ed. 2009, 48, 2.)
The concept of bioorthogonal chemical reporter has been applied to proteomic analysis of glycosylation in proteins and chemical remodeling of cell surfaces in living systems. Bioorthogonal chemical reactions have also been used for other applications, such as protein labeling, activity-based protein folding, protein targets identification, posttranslational modifications, and cell proliferation monitoring. Labeling of specific functional groups on living cell via bioorthogonal chemical reporter strategies have become increasingly powerful in cell biology. In the past few years, a tremendous progress has been made in bioorthogonal chemistry, especially that shows biocompatibility and selectivity in living systems. These approaches are often based on cycloadditions as ideal bioorthogonal reactions because of their intrinsic selectivity and tunable electronics. However, there are still many challenges facing the field, particularly from the perspective of cellular and organismal applications. For example, most bioorthogonal reporter strategies entail multistep procedures that use fluorophroe-labeled reactant partners, which often cause high background fluorescent noise that is difficult to remove from intracellular environments or tissues. In addition, these methods require high concentrations of reagents and catalysts in order to achieve detectable signals.
Some recent efforts have been focused on the design of non- or weak fluorescent probe upon CuAAC reactions with non-fluorescent alkynes or azides, which can ligate to afford a highly fluorescent triazole complex (FIG. 2). (Zhou and Fahrni, J. Am. Chem. Soc. 2004, 126, 8862; Sivakumar, et al., Org. Lett. 2004, 24, 4603; Sawa, et al., Proc. Nat. Acad. Sci. U.S.A., 2006, 103, 12371; Xie, et al., Tetrahedron 2008, 64, 2906; Li, et al., Org. Lett. 2009, 11, 3008; Le Droumaguet, et al., Chem. Soc. Rev. 2010, 39, 1223; Qi, et al, Bioconjugate Chem. 2011, 22, 1758; Chao, et al., Sci. China Chemistry 2012, 55, 125. Herner, et al., Org. Biomol. Chem. 2013, 11, 3297.) This type of CuAAC reaction occurring in high efficiency would have broad applications in the emerging field of cell biology and functional proteomics due to the distinct fluorescence properties in formation of the triazole without background fluorescent noise of the starting materials. However, these azido- and alkynyl-functionalized probes usually require excitation in the UV region and emit blue light with poor quantum yield in aqueous solution; such optical properties are not ideal for biological applications.
The distinct fluorescence enhancement induced by highly efficient CuAAC reactions would have broad applications in the emerging field of cell biology and functional proteomics (Le Droumaguet, C.; Wang, C.; Wang, Q. Chem. Soc. Rev. 2010, 39, 1233-1239; Sawa, M.; Hsu, T.-L.; Itoh, T.; Sugiyama, M.; Hanson, S. R.; Vogt, P. K.; Wong, C.-H. Proc. Natl. Acad. Sci. U.S.A 2006, 103, 12371-12376, Shie, J.-J.; Liu, Y.-C.; Lee, Y.-M.; Lim, C.; Fang, J.-M.; Wong, C.-H. J. Am. Chem. Soc. 2014, 136, 9953-9961, Hsu, T.-L.; Hanson, S. R.; Kishikawa, K.; Wang, S.-K.; Sawa, M.; Wong, C.-H. Proc. Natl. Acad. Sci. U.S.A 2007, 104, 2614-2619, Tsai, C.-S.; Liu, P.-Y.; Yen, H.-Y.; Hsu, T.-L.; Wong C.-H. Chem. Commun. 2010, 46, 5575-5577). However, the toxicity of Cu(I) has hindered the use of CuAAC in living systems.
To circumvent the cytotoxicity problem associated with the metal catalyst, the ring strain-promoted azide-alkyne cycloadditions (SPAAC) have been developed as an alternative strategy (Jewett, J. C.; Bertozzi, C. R. Chem. Soc. Rev. 2010, 39, 1272-1279, Debets, M. F.; van Berkel, S. S.; Dommerholt, J.; Dirks, A. T. J.; Rutjes, F. P. J. T.; van Delft, F. L. Acc. Chem. Res. 2011, 44, 805-815). A cyclooctyne moiety is often incorporated as a stem structure into the SPAAC reagents, such as difluorinated cyclooctynes (DIFO) and the derivatives (Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. J. Am. Chem. Soc. 2004, 126, 15046-15047, Codelli, J. A.; Baskin, J. M.; Agard, N. J.; Bertozzi, C. R. J. Am. Chem. Soc. 2008, 130, 11486-11493). To increase the ring strain, the cyclooctyne moiety can be fused with other rings to give SPAAC reagents with higher reactivity, such as dibenzylcyclooctyne (DIBO) (Ning, X.; Guo, J.; Wolfert, M. A.; Boons, G.-J. Angew. Chem. Int. Ed. 2008, 47, 2253-2255, Poloukhtine, A. A.; Mbua, N. E.; Wolfert, M. A.; Boons, G.-J.; Popik, V. V. J. Am. Chem. Soc. 2009, 131, 15769-15777, Stöckmann, H.; Neves, A. A.; Stairs, S.; Ireland-Zecchini, H.; Brindle, K. M.; Leeper, F. J. Chem. Sci. 2011, 2, 932-936, Friscourt, F.; Ledin, P. A.; Mbua, N. E.; Flanagan-Steet, H. R.; Wolfert, M. A.; Steet, R.; Boons, G.-J. J. Am. Chem. Soc. 2012, 134, 5381-5389) diarylazacyclooctynone (BARAC) (Jewett, J. C.; Sletten, E. M.; Bertozzi, C. R. J. Am. Chem. Soc. 2010, 132, 3688-3690) and bicyclononynes (BCN) (Dommerholt, J.; Schmidt, S.; Temming, R.; Hendriks, L. J. A.; Rutjes, F. P. J. T.; van Hest, J. C. M.; Lefeber, D. J.; Friedl, P.; van Delft, F. L. Angew. Chem. Int. Ed. 2010, 49, 9422-9425). Tetramethylthiacycloheptyne (TMTH) bearing a contracted seven-membered ring also exhibits reactivity in cycloaddition reactions with azides (de Almeida, G.; Sletten, E. M.; Nakamura, H.; Palaniappan, K. K.; Bertozzi, C. R. Angew. Chem. Int. Ed. 2012, 51, 2443-2447, King, M., Baati, R.; Wagner, A. Chem. Commun. 2012, 48, 9308-9309). Two cyclooctyne-based fluorogenic probes, CoumBARAC (Jewett, J. C.; Bertozzi, C. R. Org. Lett. 2011, 13, 5937-5939) and Fl-DIBO (Friscourt, F.; Fahrni, C. J.; Boons, G.-J. J. Am. Chem. Soc. 2012, 134, 18809-18815) have been described by the Bertozzi and Boons groups, respectively.
4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (also known as BODIPY) dyes are a type of popular fluorophores for many biological applications. BODIPY dyes have numerous advantages including great chemical and photophysical stability, relatively high molar absorption coefficients and fluorescence quantum yields (Φfl), excitation/emission wavelengths in the visible spectral region (longer than 500 nm), and narrow emission bandwidths with high peak intensities. (Loudet and Burgess, Chem. Rev. 2007, 107, 4891; Ulrich et al., Angew. Chem. Int. Ed. 2008, 47, 1184; Boens, et al., Chem. Soc. Rev. 2012, 41, 1130; Kamkaew, et al, Chem. Soc. Rev. 2013, 42, 77.)
Some azido-BODIPY derivatives have been developed for fluorescent labeling upon CuAAC reactions. (Li, et al., J. Org. Chem. 2008, 73, 1963.) Specifically, the low fluorescence 3-azido-BODIPY derivatives have been shown to undergo a CuAAC reaction to give the corresponding triazole with enhanced fluorescence. Although the triazole product provided a 300-fold increased emission compared to azido-BODIPY, it exhibited a low fluorescence quantum yield (Φfl<0.03) and the unreacted azido-BODIPY compound is unstable and fails to react with alkynyl biomolecules under physiological conditions, making it is incompatible with many biological applications. (Wang, et al., Sci. China Chemistry 2012, 55, 125; Chauhan, et al. Tetrahedron Lett. 2014, 55, 244.)